Enzyme preparations - sources of β-galactosidase, are widely used in the dairy industry. However, there is no standardized methods to determine their activity. Existing methods differ by the type of substrate used, conditions of analysis and how to define the products of the hydrolysis. The aim of this work is to study the process of conversion biocatalytic milk sugar and experimental substantiation of methods of analysis of hydrolysis products, providing reliable results determine activity of β-galactosidase. Found that of chemical methods glucose oxidase showed a greater sensitivity towards low concentrations of the product of hydrolysis is glucose formed in the conversion process lactose. The results of testing showed that the mass concentration of the glucose formed in hydrolysis of lactose, HPLC tested, consistent with data obtained using the glucose oxidase method. Set optimum range, which was observed directly proportional to the level of glucose concentration rise in reaction mixture from the lengthy process and the concentration of enzyme: hydrolysis time - from 10 to -6 20 min, produced glucose - from 20 μcg to 40 mcg, while the mass of enzyme - 25.0·10 , and from 40 mcg to 80 mcg, when mass - -5 5,0·10 g. When the values of absorbance of the reaction mixture ranged 0,045-0,300 to ensure reliable performance. Studies on determination of the activity of the enzyme preparations confirmed received experimental data and allowed to choose optimum parameters and conditions analysis for introducing them to the method for determining the activity β-galactosidase using glucose oxidase reagent: time of inactivation enzyme (control) -10 min. in a boiling water bath, substrate - 2% solution of lactose, pH of the substrate-6,0, hydrolysis time 15 min. at 30° c.